Neurotransmitter-gated membrane ion channels are among the most important target sites of alcohol action in the nervous system, although the manner in which alcohols modulate the function of these transmembrane proteins has not been established. The aim of this project was to investigate the actions of alcohols and related compounds on neurotransmitter-gated ion channels thought to be involved in producing the intoxicating effects of alcohols in nervous tissue. The function of the N-methyl-D-aspartate (NMDA) receptor-channel, a type of receptor for the excitatory neurotransmitter glutamate, is inhibited by intoxicating concentrations of ethanol. Results of previous studies have established that alcohols inhibit NMDA receptors via an action on gating of the ion channel, largely by decreasing mean open time, rather than by influencing agonist or coagonist binding. Using electrophysiological patch-clamp recording, we have localized the probable site of ethanol action either to the extracellular region of the protein, or to a region of one of the transmembrane domains near the extracellular surface. Using tryptophan-scanning mutagenesis, we have recently identified sites in the membrane-associated domains of the NR1 and NR2 subunits of the NMDA receptor that powerfully influence ion channel gating and affect alcohol sensitivity, and thus could be sites of alcohol action. In particular, at a position in the fourth membrane-associated domain, amino acid substitutions can produce marked changes in desensitization and mean open time. Amino acid substitutions at this position also alter ethanol sensitivity over a range that is greater than that observed for wild-type NR2 subunits. These changes in ethanol sensitivity, however, do not appear to correlate strongly with mean open time of the ion channel, but were highly correlated with both the molecular volume and hydrophobicity of the substituent. These results are consistent with a role of this position in forming a site, or part of a site, of alcohol action. Experiments in this unit have also recently identified a novel mechanism for the regulation of a neurotransmitter-gated ion channel by the ubiquitous intracellular modulatory proteins known as G proteins. In cell lines and neurons expressing receptors for the inhibitory amino acid glycine, G protein beta,gamma-subunits directly interact with an intracellular region of the receptor, enhancing its activity by increasing its affinity for glycine. This is the first demonstration that G protein beta,gamma-subunits regulate the activity of a ligand-gated ion channel.